Accurate profiling of forensic autosomal STRs using the Oxford Nanopore Technologies MinION device.

The high variability characteristic of short tandem repeat (STR) markers is harnessed for human identification in forensic genetic analyses. Despite the power and reliability of current typing techniques, sequence-level information both within and around STRs are masked in the length-based profiles generated. Forensic STR typing using next generation sequencing (NGS) has therefore gained attention as an alternative to traditional capillary electrophoresis (CE) approaches. In this proof-of-principle study, we evaluate the forensic applicability of the newest and smallest NGS platform available - the Oxford Nanopore Technologies (ONT) MinION device. Although nanopore sequencing on the handheld MinION offers numerous advantages, including low startup cost and on-site sample processing, the relatively high error rate and lack of forensic-specific analysis software has prevented accurate profiling across STR panels in previous studies. Here we present STRspy, a streamlined method capable of producing length- and sequence-based STR allele designations from noisy, error-prone third generation sequencing reads. To assess the capabilities of STRspy, seven reference samples (female: n = 2; male: n = 5) were amplified at 15 and 30 PCR cycles using the Promega PowerSeq 46GY System and sequenced on the ONT MinION device in triplicate. Basecalled reads were then processed with STRspy using a custom database containing alleles reported in the STRSeq BioProject NIST 1036 dataset. Resultant STR allele designations and flanking region single nucleotide polymorphism (SNP) calls were compared to the manufacturer-validated genotypes for each sample. STRspy generated robust and reliable genotypes across all autosomal STR loci amplified with 30 PCR cycles, achieving 100% concordance based on both length and sequence. Furthermore, we were able to identify flanking region SNPs in the 15-cycle dataset with > 90% accuracy. These results demonstrate that when analyzed with STRspy ONT reads can reveal additional variation in and around STR loci depending on read coverage. As the first and only third generation sequencing platform-specific method to successfully profile the entire panel of autosomal STRs amplified by a commercially available multiplex, STRspy significantly increases the feasibility of nanopore sequencing in forensic applications.

Differentiation of Hispanic biogeographic ancestry with 80 ancestry informative markers.

Ancestry informative single nucleotide polymorphisms (SNPs) can identify biogeographic ancestry (BGA); however, population substructure and relatively recent admixture can make differentiation difficult in heterogeneous Hispanic populations. Utilizing unrelated individuals from the Genomic Origins and Admixture in Latinos dataset (GOAL, n = 160), we designed an 80 SNP panel (Setser80) that accurately depicts BGA through STRUCTURE and PCA. We compared our Setser80 to the Seldin and Kidd panels via resampling simulations, which models data based on allele frequencies. We incorporated Admixed American 1000 Genomes populations (1000 G, n = 347), into a combined populations dataset to determine robustness. Using multinomial logistic regression (MLR), we compared the 3 panels on the combined dataset and found overall MLR classification accuracies: 93.2% Setser80, 87.9% Seldin panel, 71.4% Kidd panel. Naïve Bayesian classification had similar results on the combined dataset: 91.5% Setser80, 84.7% Seldin panel, 71.1% Kidd panel. Although Peru and Mexico were absent from panel design, we achieved high classification accuracy on the combined populations for Peru (MLR = 100%, naïve Bayes = 98%), and Mexico (MLR = 90%, naïve Bayes = 83.4%) as evidence of the portability of the Setser80. Our results indicate the Setser80 SNP panel can reliably classify BGA for individuals of presumed Hispanic origin.

Approaches to Whole Mitochondrial Genome Sequencing on the Oxford Nanopore MinION.

Traditional approaches for interrogating the mitochondrial genome often involve laborious extraction and enrichment protocols followed by Sanger sequencing. Although preparation techniques are still demanding, the advent of next-generation or massively parallel sequencing has made it possible to routinely obtain nucleotide-level data with relative ease. These short-read sequencing platforms offer deep coverage with unparalleled read accuracy in high-complexity genomic regions but encounter numerous difficulties in the low-complexity homopolymeric sequences characteristic of the mitochondrial genome. The inability to discern identical units within monomeric repeats and resolve copy-number variations for heteroplasmy detection results in suboptimal genome assemblies that ultimately complicate downstream data analysis and interpretation of biological significance. Oxford Nanopore Technologies offers the ability to generate long-read sequencing data on a pocket-sized device known as the MinION. Nanopore-based sequencing is scalable, portable, and theoretically capable of sequencing the entire mitochondrial genome in a single contig. Furthermore, the recent development of a nanopore protein with dual reader heads allows for clear identification of nucleotides within homopolymeric stretches, significantly increasing resolution throughout these regions. The unrestricted read lengths, superior homopolymeric resolution, and affordability of the MinION device make it an attractive alternative to the labor-intensive, time-consuming, and costly mainstay deep-sequencing platforms. This article describes three approaches to extract, prepare, and sequence mitochondrial DNA on the Oxford Nanopore MinION device. Two of the workflows include enrichment of mitochondrial DNA prior to sequencing, whereas the other relies on direct sequencing of native genomic DNA to allow for simultaneous assessment of the nuclear and mitochondrial genomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Enrichment-free mitochondrial DNA sequencing Alternate Protocol 1: Mitochondrial DNA sequencing following enrichment with polymerase chain reaction (PCR) Alternate Protocol 2: Mitochondrial DNA sequencing following enrichment with PCR-free hybridization capture Support Protocol 1: DNA quantification and quality assessment using the Agilent 4200 TapeStation System Support Protocol 2: AMPure XP bead clean-up Support Protocol 3: Suggested data analysis pipeline.

Nanopore sequencing: An enrichment-free alternative to mitochondrial DNA sequencing.

Mitochondrial DNA sequence data are often utilized in disease studies, conservation genetics and forensic identification. The current approaches for sequencing the full mtGenome typically require several rounds of PCR enrichment during Sanger or MPS protocols followed by fairly tedious assembly and analysis. Here we describe an efficient approach to sequencing directly from genomic DNA samples without prior enrichment or extensive library preparation steps. A comparison is made between libraries sequenced directly from native DNA and the same samples sequenced from libraries generated with nine overlapping mtDNA amplicons on the Oxford Nanopore MinION™ device. The native and amplicon library preparation methods and alternative base calling strategies were assessed to establish error rates and identify trends of discordance between the two library preparation approaches. For the complete mtGenome, 16 569 nucleotides, an overall error rate of approximately 1.00% was observed. As expected with mtDNA, the majority of error was detected in homopolymeric regions. The use of a modified basecaller that corrects for ambiguous signal in homopolymeric stretches reduced the error rate for both library preparation methods to approximately 0.30%. Our study indicates that direct mtDNA sequencing from native DNA on the MinION™ device provides comparable results to those obtained from common mtDNA sequencing methods and is a reliable alternative to approaches using PCR-enriched libraries.

Admixture Effects on Coevolved Metabolic Systems.

Oxidative phosphorylation (OXPHOS) is the primary energy generating system in eukaryotic organisms. The complexes within the OXPHOS pathway are of mixed genomic origin. Although most subunit-coding genes are located within the nuclear genome, several genes are coded for in the mitochondrial genome. There is strong evidence to support coadaptation between the two genomes in these OXPHOS gene regions in order to create tight protein interactions necessary for a functional energetics system. In this study, we begin to assess the physiological impact of separating coevolved protein motifs that make up the highly conserved energy production pathway, as we hypothesize that divergent matings will significantly diminish the protein interactions and therefore hinder efficient OXPHOS activity We measured mitochondrial activity in high energy-demanding tissues from six strains of Mus musculus with varying degrees of mixed ancestral background. Mice with divergent mitochondrial and nuclear backgrounds consistently yielded lower mitochondrial activity. Bioinformatic analysis of common single nucleotide variants across the nuclear and mitochondrial genomes failed to identify any non-synonymous variants that could account for the energetic differences, suggesting that interpopulational mating between ancestrally distinct groups influences energy production efficiency.

Cell Persistence of Allogeneic Keratinocytes and Fibroblasts Applied in a Fibrin Matrix to Acute, Full Thickness Wounds.

HP802-247 is a living cell suspension of cultured allogeneic growth-arrested human male keratinocytes and fibroblasts (1:9 ratio), intended for spray application to chronic wounds. In this study, a small wound was created on the arms of 28 healthy female volunteers (3-mm punch), followed by a single application of HP802-247. At each subsequent week for 8 weeks, a punch excision of the wounds was performed on a cohort of three subjects. Excised specimens were analyzed for allogeneic fibroblast and keratinocyte DNA determined by Y-chromosome short-tandem repeats using PCR amplification followed by capillary electrophoresis, a method with estimated sensitivity of 1 male cell in a background of 8,000 female cells. A complete haplotype attributable to HP802-247 fibroblasts was detected in three of three samples at 1 week, with one partial and one complete fibroblast haplotype detected at 2 weeks, and one partial keratinocyte haplotype detected at 3 weeks postapplication. The findings indicate that HP802-247 can be expected to persist in an acute wound bed for up to 2 weeks postapplication.

Automated analysis of sequence polymorphism in STR alleles by PCR and direct electrospray ionization mass spectrometry.

Short tandem repeats (STRs) are the primary genetic markers used for the analysis of biological samples in forensic and human identity testing. The discrimination power of a combination of STRs is sufficient in many human identity testing comparisons unless the evidence is substantially compromised and/or there are insufficient relatives or a potential mutation may have arisen in kinship analyses. An automated STR assay system that is based on electrospray ionization mass spectrometry (ESI-MS) has been developed that can increase the discrimination power of some of the CODIS core STR loci and thus provide more information in typical and challenged samples and cases. Data from the ESI-MS STR system is fully backwards compatible with existing STR typing results generated by capillary electrophoresis. In contrast, however, the ESI-MS analytical system also reveals nucleotide polymorphisms residing within the STR alleles. The presence of these polymorphisms expands the number of alleles at a locus. Population studies were performed on the 13 core CODIS STR loci from African Americans, Caucasians and Hispanics capturing both the length of the allele, as well as nucleotide variations contained within repeat motifs or flanking regions. Such additional polymorphisms were identified in 11 of the 13 loci examined whereby several nominal length alleles were subdivided. A substantial increase in heterozygosity was observed, with close to or greater than 5% of samples analyzed being heterozygous with equal-length alleles in at least one of five of the core CODIS loci. This additional polymorphism increases discrimination power significantly, whereby the seven most polymorphic STR loci have a discrimination power equivalent to the 10 most discriminating of the CODIS core loci. An analysis of substructure among the three population groups revealed a higher θ than would be observed compared with using alleles designated by nominal length, i.e., repeats solely. Two loci, D3S1358 and vWA produced θ estimates of 0.0477 and 0.0234, respectively, when the expanded allele complement (i.e., nominal allele and SNPs) was considered compared to 0.0145 and 0.01266, respectively when only nominal repeat number was considered. These differences may indicate underlying population specific allele distributions exist within these populations. A system of nomenclature has been developed that facilitates the databasing, searching and analyses of these combined data forms.

Genetic composition of six miniSTR in a Brazilian Mulatto sample population.

The present study characterizes the genetic variability of Mulatto population based on the polymorphism of six miniSTR autosomal loci, known as Non Codis 01 and 02 (NC01 and NC02) and evaluate their applicability in forensic genetics. A sample of 102 unrelated Brazilian mulattoes were genotyped for miniSTR loci D1S1677, D2S441, D4S2364 (miniplex NC02) and 45 individuals for D10S1248, D14S1434, D22S1045 (miniplex NC01). No significant deviations from Hardy-Weinberg equilibrium expectations were detected. The combined power of discrimination (PD) and mean power of exclusion (PE) were 0.999996 and 0.98991, respectively. The results also support the effectiveness of the NC01and NC02 miniplexes for human identification.

US forensic Y-chromosome short tandem repeats database.

A forensic Y-STR database generated in the US was compiled with profiles containing a portion or complete typing of 16 STR markers DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS456, DYS458, DYS635, DYS448, and Y GATA H4. There were 17,447 samples in the version of database in which 77% and 20% were collected in North America and Asia, respectively. The database was separated into six general populations, African American, Asian, Caucasian, Hispanic, Indian, and Native American. Each population was further classified into subgroups according to geographic regions. Some subgroups were tested, found to be homogenous and merged together. Allele and haplotype frequencies, as well as sample sizes were summarized. Of the full haplotypes (i.e., 16 STRs without missing data), 93.7% in total population were distinct, 92.9% were population specific, and 89.3% were only observed once. The majority of shared haplotypes were found among North American populations as a result of admixture lasting the past few hundred years. The power of discrimination (PD), coancestry coefficient (F(st)), and coefficient of gene differentiation (G(st)) at locus and haplotype levels were also calculated. The most polymorphic marker was DYS385; this marker contains a tandem duplication and actually is composed of two loci. Both G(st) and F(st) estimates were very small with haplotypes composed of a high number of STRs haplotypes (e.g., 10-16 markers), although G(st) is slightly more conservative for these extended haplotypes. With Native American removed from the total population data set, the G(st) and F(st) estimates reduce further. PD was 0.9998 for the total population dataset for all 16 Y-STR markers. Three measures of Y-STR profile frequency were calculated: (1) unconditional haplotype frequency, (2) population substructure adjusted frequency, and (3) binomial upper bound of the haplotype frequency. The binomial upper bound is the most conservative estimate for most forensic applications. Estimates of the weight of a Y-STR haplotype can be estimated using population specific or total population databases.

Haplotype block: a new type of forensic DNA markers.

Forensic DNA analysis is currently performed using highly discriminating short tandem repeat (STR) markers. SNPs are being investigated as adjunct tools for human identity testing because of their abundance in the human genome, utility for genotyping degraded DNA samples, and amenability to automation. While SNPs can provide an alternative approach, on a per locus basis they have a lower power of discrimination (PD) than STRs. With the discovery of block structures in the human genome, a novel set of SNP markers are available for further exploration of forensic utility. Several neighboring, tightly linked SNPs are inherited together and form a haplotype block, which as a haploblock has a higher discrimination power than the individual SNPs within the block. Candidate haplotype blocks were selected from three major populations (Caucasian, East Asian, and African) using the following parameters: maximum match probability reduction = 0.85, linkage disequilibrium (LD) r(2) ≥ 0.7, maximum F(st) = 0.06, minimum number of SNPs = 3, minimum heterozygosity = 0.2, and minimum number of haplotypes = 3. From the HapMap Phase II data, 253 haploblocks were identified on the 22 autosomal chromosomes. After removing haploblocks deviating from the Hardy-Weinberg equilibrium (HWE) or in LD with other haploblocks, 24 haploblocks remained as candidates for forensic consideration. The cumulative PD of these blocks can reach 10(-12) in the populations studied. The data support within and between haplotype independence even when they are syntenic. We propose guidelines for evidence interpretation that address the application of haplotype blocks for transfer evidence, mixture, and kinship analyses.

Texas population substructure and its impact on estimating the rarity of Y STR haplotypes from DNA evidence*.

Three sampled populations of unrelated males--African American, Caucasian, and Hispanic, all from Texas-were typed for 16 Y short tandem repeat (STR) markers using the AmpFlSTR Yfiler kit. These samples also were typed previously for the 13 core CODIS autosomal STR loci. Most of the 16 marker haplotypes (2478 out of 2551 distinct haplotypes) were observed only once in the data sets. Haplotype diversities were 99.88%, 99.89%, and 99.87% for the African American, Caucasian, and Hispanic sample populations, respectively. F(ST) values were very small when a haplotype comprised 10-16 markers. This suggests that inclusion of substructure correction is not required. However, haplotypes consisting of fewer loci may require the inclusion of F(ST) corrections. The testing of independence of autosomal and Y STRs supports the proposition that the frequencies of autosomal and Y STR profiles can be combined using the product rule.

Mutation rates at Y chromosome short tandem repeats in Texas populations.

Father-son pairs from three populations (African American, Caucasian, and Hispanic) of Texas were typed for the 17 Y STR markers DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS456, DYS458, DYS635, DYS448, and Y GATA H4 using the AmpFlSTR YfilerTM kit. With 49,578 allele transfers, 102 mutations were detected. One three-step and four two-step mutations were found, and all others (95.1%) were one-step mutations. The number of gains (48) and losses (54) of repeats were nearly similar. The average mutation rate in the total population is 2.1 x 10(-3) per locus (95% CI (1.7-2.5)x10(-3)). African Americans showed a higher mutation rate (3.0 x 10(-3); 95% CI (2.4-4.0)x10(-3)) than the Caucasians (1.7 x 10(-3); 95% CI (1.1-2.5)x10(-3)) and Hispanics (1.5 x 10(-3); 95% CI (1.0-2.2)x10(-3)), but grouped by repeat-lengths, such differences were not significant. Mutation is correlated with relative length of alleles, i.e., longer alleles are more likely to mutate compared with the shorter ones at the same locus. Mutation rates are also correlated with the absolute number of repeats, namely, alleles with higher number of repeats are more likely to mutate than the shorter ones (p-value=0.030). Finally, occurrences of none, one, and two mutations over the father-son transmission of alleles were consistent with the assumption of independence of mutation rates across loci.

Null allele sequence structure at the DYS448 locus and implications for profile interpretation.

Null alleles can occur with any PCR-based STR typing system. They generally are due to deletions within the target region or primer binding sites or by primer binding site mutations that destabilize hybridization of at least one of the primers flanking the target region. Although not common, null types were detected at the DYS448 locus in seven out of 1,005 unrelated males in the Hispanic population. Of these DYS448 null types, four individuals displayed an apparent duplication at the DYS437 locus. The additional allele observed at the DYS437 locus is in actuality a smaller-sized DYS448 amplicon, which is the result of a deletion of the invariant N42 base pair domain and downstream repeats within the DYS448 locus. Thus, some DYS448 null types are not truly null. A true DYS448 null allele carried numerous primer binding site variants and a large deletion including the N42 base pair domain and surrounding or downstream repeat regions. The presence of null alleles is not a real concern for interpretation of Y STR loci evidence; current methods for interpreting Y STR profiles easily accommodate such phenomena.